| 11. |
- Nikpour, Mahnaz, 1980, et al.
(författare)
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Proteoglycan profiling of human, rat and mouse insulin-secreting cells
- 2021
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 31:8, s. 916-930
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycans (PGs) are proteins with glycosaminoglycan (GAG) chains, such as chondroitin sulfate (CS) or heparan sulfate (HS), attached to serine residues. We have earlier shown that prohormones can carry CS, constituting a novel class of PGs. The mapping of GAG modifications of proteins in endocrine cells may thus assist us in delineating possible roles of PGs in endocrine cellular physiology. With this aim, we applied a glycoproteomic approach to identify PGs, their GAG chains and their attachment sites in insulin-secreting cells. Glycopeptides carrying GAG chains were enriched from human pancreatic islets, rat (INS-1 832/13) and mouse (MIN6, NIT-1) insulinoma cell lines by exchange chromatography, depolymerized with GAG lyases, and analyzed by nanoflow liquid chromatography tandem mass spectrometry. We identified CS modifications of chromogranin-A (CgA), islet amyloid polypeptide, secretogranin-1 and secretogranin-2, immunoglobulin superfamily member 10, and protein AMBP. Additionally, we identified two HS-modified prohormones (CgA and secretogranin-1), which was surprising, as prohormones are not typically regarded as HSPGs. For CgA, the glycosylation site carried either CS or HS, making it a so-called hybrid site. Additional HS sites were found on syndecan-1, syndecan-4, nerurexin-2, protein NDNF and testican-1. These results demonstrate that several prohormones, and other constituents of the insulin-secreting cells are PGs. Cell-targeted mapping of the GAG glycoproteome forms an important basis for better understanding of endocrine cellular physiology, and the novel CS and HS sites presented here provide important knowledge for future studies.
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| 12. |
- Nilsson, Jonas, 1970, et al.
(författare)
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A glycomic workflow for LC-MS/MS analysis of urine glycosaminoglycan biomarkers in mucopolysaccharidoses
- 2023
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Ingår i: Glycoconjugate Journal. - 0282-0080. ; 40:5, s. 523-40
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Tidskriftsartikel (refereegranskat)abstract
- In recent years, several rational designed therapies have been developed for treatment of mucopolysaccharidoses (MPS), a group of inherited metabolic disorders in which glycosaminoglycans (GAGs) are accumulated in various tissues and organs. Thus, improved disease-specific biomarkers for diagnosis and monitoring treatment efficacy are of paramount importance. Specific non-reducing end GAG structures (GAG-NREs) have become promising biomarkers for MPS, as the compositions of the GAG-NREs depend on the nature of the lysosomal enzyme deficiency, thereby creating a specific pattern for each subgroup. However, there is yet no straightforward clinical laboratory platform which can assay all MPS-related GAG-NREs in one single analysis. Here, we developed and applied a GAG domain mapping approach for analyses of urine samples of ten MPS patients with various MPS diagnoses and corresponding aged-matched controls. We describe a nano-LC-MS/MS method of GAG-NRE profiling, utilizing 2-aminobenzamide reductive amination labeling to improve the sensitivity and the chromatographic resolution. Diagnostic urinary GAG-NREs were identified for MPS types IH/IS, II, IIIc, IVa and VI, corroborating GAG-NRE as biomarkers for these known enzyme deficiencies. Furthermore, a significant reduction of diagnostic urinary GAG-NREs in MPS IH (n = 2) and MPS VI (n = 1) patients under treatment was demonstrated. We argue that this straightforward glycomic workflow, designed for the clinical analysis of MPS-related GAG-NREs in one single analysis, will be of value for expanding the use of GAG-NREs as biomarkers for MPS diagnosis and treatment monitoring.
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| 13. |
- Nilsson, Jonas, 1970, et al.
(författare)
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Expression of influenza A virus glycan receptor candidates in mallard, chicken, and tufted duck
- 2024
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Ingår i: GLYCOBIOLOGY. - 0959-6658 .- 1460-2423. ; 34:3
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Tidskriftsartikel (refereegranskat)abstract
- Influenza A virus (IAV) pandemics result from interspecies transmission events within the avian reservoir and further into mammals including humans. Receptor incompatibility due to differently expressed glycan structures between species has been suggested to limit zoonotic IAV transmission from the wild bird reservoir as well as between different bird species. Using glycoproteomics, we have studied the repertoires of expressed glycan structures with focus on putative sialic acid-containing glycan receptors for IAV in mallard, chicken and tufted duck; three bird species with different roles in the zoonotic ecology of IAV. The methodology used pinpoints specific glycan structures to specific glycosylation sites of identified glycoproteins and was also used to successfully discriminate alpha 2-3- from alpha 2-6-linked terminal sialic acids by careful analysis of oxonium ions released from glycopeptides in tandem MS/MS (MS2), and MS/MS/MS (MS3). Our analysis clearly demonstrated that all three bird species can produce complex N-glycans including alpha 2-3-linked sialyl Lewis structures, as well as both N- and O- glycans terminated with both alpha 2-3- and alpha 2-6-linked Neu5Ac. We also found the recently identified putative IAV receptor structures, Man-6P N-glycopeptides, in all tissues of the three bird species. Furthermore, we found many similarities in the repertoires of expressed receptors both between the bird species investigated and to previously published data from pigs and humans. Our findings of sialylated glycan structures, previously anticipated to be mammalian specific, in all three bird species may have major implications for our understanding of the role of receptor incompatibility in interspecies transmission of IAV.
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| 14. |
- Nilsson, Jonas, 1970, et al.
(författare)
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N-glycoproteomic analyses of human intestinal enteroids, varying in histo-blood group geno- and phenotypes, reveal a wide repertoire of fucosylated glycoproteins
- 2024
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Ingår i: GLYCOBIOLOGY. - 0959-6658 .- 1460-2423. ; 34:6
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Tidskriftsartikel (refereegranskat)abstract
- Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.
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| 15. |
- Noborn, Fredrik, et al.
(författare)
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A Glycoproteomic Approach to Identify Novel Proteoglycans
- 2022
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Ingår i: Glycosaminoglycans. Methods in Molecular Biology, vol 2303. Balagurunathan K., Nakato H., Desai U., Saijoh Y. (eds). - New York, NY : Springer. - 1064-3745. - 9781071613986 ; , s. 71-85
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Bokkapitel (refereegranskat)abstract
- In this chapter, we describe a glycoproteomic approach for the identification of novel chondroitin sulfate proteoglycans (CSPGs) using a combination of biochemical enrichments, enzymatic digestions, and nanoscale liquid chromatography tandem mass spectrometry (nLC-MS/MS) analysis. The identification is achieved by trypsin digestion of CSPG-containing samples, followed by enrichment of chondroitin sulfate (CS) glycopeptides by strong anion exchange chromatography (SAX). The enriched CS glycopeptides are then digested with chondroitinase ABC to depolymerize the CS polysaccharides, generating a residual hexasaccharide structure, composed of the linkage region tetrasaccharide extended with a terminal dehydrated disaccharide, still attached to the peptide. The obtained CS glycopeptides are analyzed by nLC-MS/MS, and the generated data sets are evaluated through proteomic software with adjustment in the settings to allow for glycopeptide identification. This approach has enabled the identification of several novel core proteins in human samples and in Caenorhabditis elegans. Here we specifically describe the procedure for the enrichment and characterization of CS glycopeptides from human cerebrospinal fluid (CSF). © 2022, Springer Science+Business Media, LLC, part of Springer Nature.
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| 16. |
- Noborn, Fredrik, et al.
(författare)
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Characterization of C. elegans Chondroitin Proteoglycans and Their Large Functional and Structural Heterogeneity; Evolutionary Aspects on Structural Differences Between Humans and the Nematode.
- 2021
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Ingår i: Advances in experimental medicine and biology. - Cham : Springer International Publishing. - 0065-2598. ; 21, s. 155-170
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Forskningsöversikt (refereegranskat)abstract
- Proteoglycans regulate important cellular pathways in essentially all metazoan organisms. While considerable effort has been devoted to study structural and functional aspects of proteoglycans in vertebrates, the knowledge of the core proteins and proteoglycan-related functions in invertebrates is relatively scarce, even for C.elegans. This nematode produces a large amount of non-sulfated chondroitin in addition to small amount of low-sulfated chondroitin chains (Chn and CS chains, respectively). Until recently, 9 chondroitin core proteins (CPGs) had been identified in C.elegans, none of which showed any homology to vertebrate counterparts or to other invertebrate core proteins. By using a glycoproteomic approach, we recently characterized the chondroitin glycoproteome of C.elegans, resulting in the identification of 15 novel CPG core proteins in addition to the 9 previously established. Three of the novel core proteins displayed homology to human proteins, indicating that CPG and CSPG core proteins may be more conserved throughout evolution than previously perceived. Bioinformatic analysis of the primary amino acid sequences revealed that the core proteins contained a broad range of functional domains, indicating that specialization of proteoglycan-mediated functions may have evolved early in metazoan evolution. This review specifically discusses our recent data in relation to previous knowledge of core proteins and GAG-attachment sites in Chn and CS proteoglycans of C.elegans and humans, and point out both converging and diverging aspects of proteoglycan evolution.
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| 17. |
- Noborn, Fredrik, et al.
(författare)
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Expanding the Chondroitin Sulfate Glycoproteome - But How Far?
- 2021
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Ingår i: Frontiers in cell and developmental biology. - : Frontiers Media SA. - 2296-634X. ; 9
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Forskningsöversikt (refereegranskat)abstract
- Chondroitin sulfate proteoglycans (CSPGs) are found at cell surfaces and in connective tissues, where they interact with a multitude of proteins involved in various pathophysiological processes. From a methodological perspective, the identification of CSPGs is challenging, as the identification requires the combined sequencing of specific core proteins, together with the characterization of the CS polysaccharide modification(s). According to the current notion of CSPGs, they are often considered in relation to a functional role in which a given proteoglycan regulates a specific function in cellular physiology. Recent advances in glycoproteomic methods have, however, enabled the identification of numerous novel chondroitin sulfate core proteins, and their glycosaminoglycan attachment sites, in humans and in various animal models. In addition, these methods have revealed unexpected structural complexity even in the linkage regions. These findings indicate that the number and structural complexity of CSPGs are much greater than previously perceived. In light of these findings, the prospect of finding additional CSPGs, using improved methods for structural and functional characterizations, and studying novel sample matrices in humans and in animal models is discussed. Further, as many of the novel CSPGs are found in low abundance and with not yet assigned functions, these findings may challenge the traditional notion of defining proteoglycans. Therefore, the concept of proteoglycans is considered, discussing whether "a proteoglycan" should be defined mainly on the basis of an assigned function or on the structural evidence of its existence.
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| 18. |
- Noborn, Fredrik, et al.
(författare)
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Mapping the Human Chondroitin Sulfate Glycoproteome Reveals an Unexpected Correlation Between Glycan Sulfation and Attachment Site Characteristics. : Chondroitin Sulfation and Attachment Site Characteristics
- 2023
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Ingår i: Molecular & cellular proteomics : MCP. - : Elsevier. - 1535-9484 .- 1535-9476. ; 22:8
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Tidskriftsartikel (refereegranskat)abstract
- Chondroitin sulfate proteoglycans (CSPGs) control key events in human health and disease and are composed of chondroitin sulfate (CS) polysaccharide(s) attached to different core proteins. Detailed information on the biological effects of site-specific CS structures is scarce as the polysaccharides are typically released from their core proteins prior to analysis. Here we present a novel glycoproteomic approach for site-specific sequencing of CS modifications from human urine. Software-assisted and manual analysis revealed that certain core proteins carried CS with abundant sulfate modifications, while others carried CS with lower levels of sulfation. Inspection of the amino acid sequences surrounding the attachment sites indicated that the acidity of the attachment site motifs increased the levels of CS sulfation, and statistical analysis confirmed this relationship. However, not only the acidity but also the sequence and characteristics of specific amino acids in the proximity of the serine glycosylation site correlated with the degree of sulfation. These results demonstrate attachment site-specific characteristics of CS polysaccharides of CSPGs in human urine and indicate that this novel method may assist in elucidating the biosynthesis and functional roles of CSPGs in cellular physiology.
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| 19. |
- Noborn, Fredrik, et al.
(författare)
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Site-specific glycosylation of proteoglycans: A revisited frontier in proteoglycan research
- 2022
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Ingår i: Matrix Biology. - : Elsevier BV. - 0945-053X. ; 111, s. 289-306
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycans (PGs), a class of carbohydrate-modified proteins, are present in essentially all metazoan organisms investigated to date. PGs are composed of glycosaminoglycan (GAG) chains attached to various core proteins and are important for embryogenesis and normal homeostasis. PGs exert many of their func-tions via their GAG chains and understanding the details of GAG-ligand interactions has been an essential part of PG research. Although PGs are also involved in many diseases, the number of GAG-related drugs used in the clinic is yet very limited, indicating a lack of detailed structure-function understanding. Structural analysis of PGs has traditionally been obtained by first separating the GAG chains from the core proteins, after which the two components are analyzed separately. While this strategy greatly facilitates the analysis, it precludes site-specific information and introduces either a "GAG" or a "core protein" perspective on the data interpretation. Mass-spectrometric (MS) glycoproteomic approaches have recently been introduced, provid-ing site-specific information on PGs. Such methods have revealed a previously unknown structural complex-ity of the GAG linkage regions and resulted in identification of several novel CSPGs and HSPGs in humans and in model organisms, thereby expanding our view on PG complexity. In light of these findings, we discuss here if the use of such MS-based techniques, in combination with various functional assays, can also be used to expand our functional understanding of PGs. We have also summarized the site-specific information of all human PGs known to date, providing a theoretical framework for future studies on site-specific func-tional analysis of PGs in human pathophysiology.(c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
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| 20. |
- Persson, Andrea, et al.
(författare)
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Domain Mapping of Chondroitin/Dermatan Sulfate Glycosaminoglycans Enables Structural Characterization of Proteoglycans
- 2021
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Ingår i: Molecular & Cellular Proteomics. - : Elsevier BV. - 1535-9476. ; 20
-
Tidskriftsartikel (refereegranskat)abstract
- Of all posttranslational modifications known, glycosaminoglycans (GAGs) remain one of the most challenging to study, and despite the recent years of advancement in MS technologies and bioinformatics, detailed knowledge about the complete structures of GAGs as part of proteoglycans (PGs) is limited. To address this issue, we have developed a protocol to study PG-derived GAGs. Chondroitin/dermatan sulfate conjugates from the rat insulinoma cell line, INS-1832/13, known to produce primarily the PG chromogranin-A, were enriched by anion-exchange chromatography after pronase digestion. Following benzonase and hyaluronidase digestions, included in the sample preparation due to the apparent interference from oligonucleotides and hyaluronic acid in the analysis, the GAGs were orthogonally depolymerized and analyzed using nano-flow reversed-phase LC-MS/MS in negative mode. To facilitate the data interpretation, we applied an automated LC-MS peak detection and intensity measurement via the Proteome Discoverer software. This approach effectively provided a detailed structural description of the nonreducing end, internal, and linkage region domains of the CS/DS of chromogranin-A. The copolymeric CS/DS GAGs constituted primarily consecutive glucuronic-acid-containing disaccharide units, or CS motifs, of which the N-acetylgalactosamine residues were 4-0-sulfated, interspersed by single iduronic-acid-containing disaccharide units. Our data suggest a certain heterogeneity of the GAGs due to the identification of not only CS/DS GAGs but also of GAGs entirely of CS character. The presented protocol allows for the detailed characterization of PG-derived GAGs, which may greatly increase the knowledge about GAG structures in general and eventually lead to better understanding of how GAG structures are related to biological functions.
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